Preparation of potent macrophage activating factors derived from cloned vitamin D binding protein and its domain and their therapeutic usage for cancer, HIV-infection and osteopetrosis

ABSTRACT

Vitamin D-binding protein (Gc protein) and its small domain (approximately ⅕ of the Gc peptide also known as domain III) were cloned via a baculovirus vector. The cloned Gc protein and the cloned domain (Cd) peptide were treated with immobilized β-galactosidase and sialidase to yield macrophage activating factors, GcMAFc and CdMAF, respectively. These cloned macrophage activating factors and GcMAF are to be used for therapy of cancer, HIV-infection and osteopetrosis, and may also be used as adjuvants for immunization and vaccination.

FIELD OF THE INVENTION

[0001] This invention relates to potent macrophage activating factors,prepared by oligosaccharide digestion of the cloned vitamin D bindingprotein (Gc protein) and the cloned Gc protein domain III, and the useof these macrophage activating factors for various cancer, HIV-infectionand osteopetrosis, and as adjuvants for immunization and vaccination.

TABLE OF TERMS

[0002] Gc protein Vitamin D₃-binding protein

[0003] MAF macrophage activating factor

[0004] GcMAF Gc protein-derived macrophage activating protein

[0005] GcMAFc cloned Gc protein-derived macrophage activating factor

[0006] Gc domain III domain III region of Gc protein

[0007] CdMAF cloned domain III-derived macrophage activating factor

SUMMARY OF THE INVENTION

[0008] Vitamin D-binding protein (Gc protein) and its small domain(approximately ⅕ of the Gc peptide also known as domain III) were clonedvia a baculovirus vector. The cloned Gc protein and the cloned domain(Cd) peptide were treated with immobilized β-galactosidase and sialidaseto yield macrophage activating factors, GcMAFc and CdMAF, respectively.These cloned macrophage activating factors and GcMAF are to be used fortherapy of cancer, HIV-infection and osteopetrosis, and may also be usedas adjuvants for immunization and vaccination.

DESCRIPTION OF THE DRAWINGS

[0009] Other objects and many attendant features of this invention willbecome readily appreciated as the same becomes better understood byreference to the following detailed description when considered inconnection with the accompanying drawings wherein:

[0010]FIG. 1a is a schematic illustration of the formation of macrophageactivating factor (MAF).

[0011]FIG. 1b is a schematic illustration of the deglycosylation of Gcprotein in a cancer or HIV-infected patient's blood stream.

[0012]FIG. 2 shows the correlation between plasmaα-N-acetylgalactosaminidase activity and tumor burden (total cellcounts) in the peritoneal cavity of Ehrlich ascites tumor.

[0013]FIG. 3 shows the amino acid sequence of cloned GcMAF which is SEQID NO:1 which is the entire cloned Gc protein.

[0014]FIG. 4 shows the construction of the DNA fragment encoding theleader sequence of EcoRi fragment E1 and domain III regions of the Gcprotein; A, the entire cDNA for Gc protein; B, the construct to beinserted into the non-fusion vector; the shaded area indicates thecompressed regions of about 1,000 base pairs (bp).

[0015]FIG. 5 shows the 89 amino acid sequence, SEQ ID NO:2, of thecloned domain III (CdMAF₁), using the non-fusion vector.

[0016]FIG. 6 shows the baculovirus fusion vector for cloning the domainIII of Gc protein.

[0017]FIG. 7 shows the 94 amino acid sequence, SEQ ID NO:3, of thecloned domain III (CdMAF₂), using the fusion vector.

[0018]FIG. 8A shows the therapeutic effect of GcMAF in accordance withthe present invention on adult persons suffering from prostate cancer.

[0019]FIG. 8B shows the therapeutic effect of GcMAF in accordance withthe present invention on adult persons suffering from breast cancer.

[0020]FIG. 8C shows the therapeutic effect of GcMAF in accordance withthe present invention on adult persons suffering from colon cancer.

[0021]FIG. 8D shows the therapeutic effect of GcMAF in accordance withthe present invention on adult persons suffering from leukemia.

BACKGROUND OF THE INVENTION

[0022] A. Inflammatory Response Results in Activation of Macrophages

[0023] Inflammation results in the activation of macrophages. Inflamedlesions release lysophospholipids. The administration into mice of smalldoses (5-20 μg/mouse) of lysophosphatidylcholine (lyso-Pc) and otherlysophospholipids induced a greatly enhanced phagocytic and superoxidegenerating capacity of macrophages (Ngwenya and Yamamoto, Proc. Soc.Exp. Biol. Med. 193:118, 1990; Yamamoto et al, Inf. Imm. 61:5388, 1993;Yamamoto et al., Inflammation. 18:311, 1994).

[0024] This macrophage activation requires participation of B and Tlymphocytes and serum vitamin D binding protein (DBP; human DBP is knownas Gc protein). In vitro activation of mouse peritoneal macrophages bylyso-Pc requires the step-wise modification of Gc protein by3-galactosidase of Iyso-Pc-treated B cells and sialidase of T cells togenerate the macrophage activating factor (MAF), a protein withN-acetylgalactosamine as the remaining sugar moiety (FIG. 1a (Yamamotoet al., Proc. Natl. Acad. Sci. USA. 88:8539, 1991; Yamamoto et al., J.Immunol. 151:2794, 1993; Naraparaju and Yamamoto, Immunol. Lett. 43:143,1994). Thus, Gc protein is a precursor for MAF.

[0025] Incubation of Gc protein with immobilized β-galactosidase andsialidase generates a remarkably high titered MAF (GcMAF) (Yamamoto etal., Proc. Natl. Acad. Sci. USA. 88:8539, 1991; Yamamoto et al., J.Immunol. 151:2794, 1993; Naraparaju and Yamamoto, Immunol. Lett. 43:143,1994; U.S. Pat. No. 51,177,002). Administration of a minute amount (10pg/mouse; 100 ng/human) of GcMAF resulted in greatly enhanced phagocyticand super-oxide generating capacities of macrophages.

[0026] When peripheral blood monocytesimacrophages (designated asmacrophages hereafter) of 258 cancer patients bearing various types ofcancer were treated in vitro with 100 pg GcMAF/ml, macrophages of allcancer patients were activated for phagocytic and superoxide generatingcapacity. This observation indicates that cancer patient macrophages arecapable of being activated. However, the MAF precursor activity ofplasma Gc protein was lost or reduced in approximately 70% of thiscancer patient population. Loss of the MAF precursor activity preventsgeneration of MAF. Therefore, macrophage activation cannot develop incertain cancer patients. Since macrophage activation is the first stepin the immune development cascade, such cancer patients becomeimmunosuppressed. This may explain at least in part why cancer patientsdie from overwhelming infection. Lost or reduced precursor activity ofGc protein was found to be due to deglycosylation of plasma Gc proteinby α-N-acetylgalactosaminidase detected in cancer patient blood stream.Deglycosylated Gc protein cannot be converted to MAF (FIG. 1b.

[0027] Similarly, when peripheral blood macrophages of 160HIV-infected/AIDS patients were treated in vitro with 100 pg GcMAF/ml,macrophages of all patients were activated for phagocytic and superoxidegenerating capacity. However, the MAF precursor activity of plasma Gcprotein was low in approximately 35% of the HIV-infected patientpopulation. As in cancer patients, these patients' plasma Gc protein isdeglycosylated by α-N-acetylgalactosaminidase detected in HIV-infectedpatients.

[0028] Both cancer and HIV-infected patients having severely decreasedprecursor activity of plasma Gc protein carried large amounts ofα-N-acetylgalactosaminidase while patients having moderately decreasedprecursor activity had moderate levels of plasmaα-N-acetylgalactosaminidase activities. Patients with high precursoractivity, including asymptomatic HIV-infected patients, had low butsignificant levels of plasma α-N-acetylgalactosaminidase activity. Sincea large amount (260 μg/ml) of Gc protein exists in the blood stream, alow level of the enzyme does not affect the precursor activity.Nevertheless, α-N-acetylgalactosaminidase activity was found in plasmasof all cancer and HIV-infected patients and had an inverse correlationwith the precursor activity of their plasma Gc protein (Yamamoto et al.,AIDS Res. Human Ret. 11:1373, 1995). Thus, increase in patient plasmaα-N-acetylgalactosaminidase activity is responsible for decrease in theprecursor activity of plasma Gc protein. These observations lead us topropose that plasma α-N-acetylgalactosaminidase plays a role inimmunosuppression in cancer and HIV-infected/AIDS patients.

[0029] B. The Origin of Immunosuppression

[0030] The source of the plasma α-N-acetylgalactosaminidase in cancerpatients appeared to be cancerous cells. Highα-N-acetylgalactosaminidase activities were detected in tumor tissuehomogenates of various organs, including eleven different tumor tissuesincluding 4 lung, 3 breast, 3 colon and 1 cervix tumors, though theα-N-acetylgalactosaminidase activity varied from 15.9 to 50.8nmoles/mg/min. Surgical removal of malignant lesions in human cancerresults in subtle decrease of plasma α-N-acetylgalactosaminidaseactivity with concomitant increase in the precursor activity,particularly if malignant cells are localized.

[0031] In a preclinical mouse tumor model, BALB/c mice were transplantedwith 5×10⁵ Ehrlich ascites tumor cells/mice into peritoneal cavity andanalyzed for serum α-N-acetylgalactosaminidase activity. When plasmaenzyme level were measured as transplanted Ehrlich ascites tumor grew inmouse peritoneal cavity, the enzyme activity was directly proportionalto tumor burden as shown in FIG. 2. This was also confirmed with nudemouse transplanted with KB cells (human oral squamous cell carcinomacell line). Serum α-N-acetylgalactosaminidase activity increased astumor size (measured by weight) of the solid tumor increased. Thus, Ihave been using plasma α-N-acetylgalactosaminidase activity as aprognostic index to monitor the progress of therapy.

[0032] Radiation therapy of human cancer decreased plasmaα-N-acetylgalactosaminidase activity with a concomitant increase ofprecursor activity. This implies that radiation therapy decreases thenumber of cancerous cells capable of secretingα-N-acetylgalactosaminidase. These results also confirmed that plasmaα-N-acetylgalactosaminidase activity has an inverse correlation with theMAF precursor activity of Gc protein. Even after surgical removal oftumor lesions in cancer patients, most post-operative patients carriedsignificant amounts of α-N-acetylgalactosaminidase activity in theirblood stream. The remnant cancerous lesions in these post-operativepatients cannot be detectable by any other procedures, e.g., X-ray,scintigraphy, etc. I have been using this most sensitive enzyme assay asprognostic index during the course of GcMAF therapy for treating cancer.

[0033] HIV-infected cells appeared to secreteα-N-acetylgalactosaminidase. When peripheral blood mononuclear cells(PBMC) of HIV-infected patients were cultured and treated with mitomycinas a provirus inducing agent (Sato et al., Arch. Virol. 54:333, 1977),α-N-acetylgalactosaminidase was secreted into culture media. Theseresults led us to suggest that α-N-acetylgalactosaminidase is a viruscoded product. In fact, HIV-envelope protein gp120 appears to carry theα-N-acetylgalactosaminidase activity.

[0034] C. A Defect in Macrophage Activation Cascade ManifestsOsteopetrosis

[0035] An inflammation-primed macrophage activation cascade has beendefined as a major process leading to the production of macrophageactivating factor. Activation of other phagocytes such as osteoclastsshares the macrophage activation cascade (Yamamoto et al., J. Immunol.152:5100, 1994). Thus, a defect in the macrophage activation cascaderesults in lack of activation in osteoclasts.

[0036] Autosomal recessive osteopetrosis is characterized by an excessaccumulation of bone throughout the skeleton as a result ofdysfunctional osteoclasts, resulting in reduced bone resorption (Marks,Clin. Orthop. 189:239, 1984). In animal models of osteopetrosis,depending on the degree of osteoclast dysfunction, marrow cavitydevelopment and tooth eruption are either delayed or more commonlyabsent (Marks, Am. J. Med. Genet. 34:43, 1989). In human infantileosteopetrosis, death occurs within the first decade of life usuallyoverwhelming infection (Reeves, Pediatrics. 64:202,1979), indicatingimmunosuppression. Accumulated evidence suggests that deficient ordysfunctional osteoclasts in osteopetrotic animals are often accompaniedby deficiencies or dysfunctions of macrophages. The studies of thepresent inventor on the activation of both osteoclasts and macrophagesin the osteopetrotic mutations revealed that osteoclasts and macrophagescan be activated by a common signaling factor, the macrophage activatingfactor and that a defect in β-galactosidase of B cells incapacitates thegeneration process of macrophage activating factor (Yamamoto et al., J.Immunol. 152:5100, 1994). Since GcMAF and its cloned derivatives bypassthe function of lymphocytes and Gc protein and act directly onmacrophages and osteoclasts, administration of these factors intoosteopetrotic hosts should rectify the bone disorder. In fact thepresent inventor has recently found that four administrations ofpurified cloned human macrophage activating factor (GcMAFc) (100pg/week) to the p mutant mice beginning at birth for four weeks resultedin the activation of both macrophages and -osteoclasts and subsequentresorption of the excess skeletal matrix.

[0037] D. Therapeutic Application of GcMAF and its Cloned Derivatives onCancer

[0038] Despite defects in the macrophage activation cascade in cancer,HIV-infected and osteopetrotic patients, GcMAF bypasses the functions oflymphocytes and Gc protein and acts directly on macrophages (orosteoclasts) for activation. Macrophages have a potential to eliminatecancerous cells and HIV-infected cells when activated. When cancerpatients were treated with 100 ng GcMAF/patient weekly for severalmonths, GcMAF showed remarkable curative effects on a variety of humancancer indiscriminately.

[0039] Instead of obtaining of GcMAF from human blood source, it can beobtained from the cloned Gc protein or its small domain responsible formacrophage activation. The cloning Gc protein require an eukaryoticvector/host capable of the glycosylation of the cloned products. The Gcprotein having a molecular weight of 52,000 and 458 amino acid residues)is a multi-functional protein and carries three distinct domains (Cookeand Haddad, Endocrine Rev., 10:294,1989).

[0040] Domain I interacts with vitamin D while domain III interacts withactin (Haddad et al., Biochem., 31:7174, 1992). Chemically andproteolytically fragmented Gc enabled me to indicate that the smallestdomain, domain ill, contains an essential peptide for macrophageactivation. Accordingly, I cloned both Gc protein and the entire domainIII peptide, by the use of a baculovirus vector and an insect host, andtreated them with the immobilized β-galactosidase and sialidase to yieldpotent macrophage activating factors, designated GcMAFc and CdMAF,respectively. Like GcMAF, these cloned GcMAFc and CdMAF appear to havecurative effects on cancer.

[0041] E. A Potent Adjuvant Activity of GcMAF for Immunization withAntigens or Vaccines

[0042] Macrophages are antigen presenting cells. Macrophages activatedby GcMAF rapidly phagocytize target antigens or cells and presented theprocessed antigens to antibody producing cells. I observed a rapiddevelopment of a large amount of antibody secreting cells immediately (1to 4 days) after inoculation of small amount of GcMAF (100 pg/mouse) andsheep erythrocytes (SRBC). This finding indicates that GcMAF and itscloned derivatives, GcMAFc and CdMAF, should serve as potent adjuvantsfor immunization and vaccination.

DESCRIPTION OF THE METHODS FOR GENE CLONING FOR MACROPHAGE ACTIVATINGFACTORS

[0043] A. Cloning of the cDNA of Gc Protein into an Insect Virus.

[0044] A full length cDNA encoding the human Gc protein was isolatedfrom a human liver cDNA library in bacteriophage λgt11 (Clontech, PaloAlto, Calif.) by the use of pico Blue™ immunoscreening kit availablefrom Stratagene of La Jolla, Calif. The baculoviral expression system inthe insect cells takes advantages of several facts about the polyhedronprotein: (a) it is expressed to very high levels in infected cells whereit constitutes more than half of the total cellular protein late in theinfection cycle; (b) it is nonessential for infection or replication ofthe virus, meaning that the recombinant virus does not require anyhelper function; (c) viruses lacking polyhedron gene have distinctplaque morphology from viruses containing the cloned gene; and d) unlikebacterial cells, the insect cell efficiently glycosylate the cloned geneproducts.

[0045] One of the beauties of this expression system is a visual screenallowing recombinant viruses to be distinguished and quantified. Thepolyhedron protein is produced at very high levels in the nuclei ofinfected cells late in the viral infection cycle. Accumulated polyhedronprotein forms occlusion bodies that also contain embedded virusparticles. These occlusion bodies, up to 15 μm in size, are highlyrefractile, giving them a bright shiny appearance that is readilyvisualized under a light microscope. Cells infected with recombinantviruses lack occlusion bodies. To distinguish recombinant virus fromwild-type virus, the transfection supernatant (recombinant containingvirus lysate) is plaqued onto a monolayer of insect cells. Plaques arethen screened under a light microscope for the presence (indicative ofwild-type virus) or absence (indicative of recombinant virus) ofocclusion bodies.

[0046] Unlike bacterial expression systems, the baculovirus-based systemis an eukaryotic expression system and thus uses many of the proteinmodification, processing such as glycosylation, and transport reactionspresent in higher eukaryotic cells. In addition, the baculoviralexpression system uses a helper-independent virus that can be propagatedto high titers in insect cells adapted for growth in suspensioncultures, making it possible to obtain large amounts of recombinantprotein with relative ease. The majority of the overproduced proteinremains soluble in insect cells by contrast with the insoluble proteinsoften obtained from bacteria. Furthermore, the viral genome is large(130 kbp) and thus can accommodate large segments of foreign DNA.Finally, baculoviruses are noninfectious to vertebrates, and theirpromoters have been shown to be inactive in mammalian cells (Carbonellet al., J. Virol. 56:153, 1985), which gives them a possible advantageover other systems when expressing oncogenes or potentially toxicproteins.

[0047] 1) Choice of Baculoviral Vector.

[0048] All available baculoviral vectors are pUC-based and conferampicillin resistance. Each contains the polyhedron gene promoter,variable lengths of polyhedron coding sequence, and insertion site(s)for cloning the foreign gene of interest flanked by viral sequences thatlie 5′ to the promoter and 3′ to the foreign gene insert. These flankingsequences facilitate homologous recombination between the vector andwild-type baculoviral DNA (Ausubel et al., Current Protocols in Mol.Biol. 1990). The major consideration when choosing the appropriatebaculoviral expression vector is whether to express the recombinant as afusion or non-fusion protein. Since glycosylation of Gc peptide requiresa leader signal sequence for transfer of the peptide into theendoplasmic reticulum, the cDNA containing initiation codon (−16 Met)through the leader sequence to the +1 amino acid (leu) of the native Gcprotein should be introduced to non-fusion vector with a polylinkercarrying the EcoRI site, pLV1393 (Invitrogen, San Diego, Calif.).

[0049] During partial digestion of the cDNA for Go protein in λgt11 withEcoRI enzyme, a full length Gc cDNA with EcoRi termini was isolatedelectrophoretically, mixed with EcoRI-cut pVL1393, and ligated with T4ligase. This construct in correct orientation should express the entireGc peptide, a total of 458 amino acids (FIG. 3). To obtain the correctconstruction, competent E. coli HB101 cells were transformed with pVLvector and selected for transformants on Luria broth agar platescontaining ampicillin (LB/ampicillin plates). The DNA was prepared forthe sequencing procedure to determine which colony contains the insertor gene with proper reading orientation, by first searching for the 3′poly A stretch. The clones with 3′ ply A (from the poly A tail of mRNA)were then sequenced from the 5′ end to confirm the correct orientationof the full length DNA for the Gc peptide.

[0050] 2) Co-transfection of Insect Cells with the Cloned Plasmid DNAand Wild-type Viral DNA

[0051] A monolayer (2.5×10⁶ cells in each of 25-cm² flasks) ofSpodoptera frugiperda (Sf9) cells was co-transfected with a clonedplasmid (vector) DNA (2 μg) and a wild-type (AcMNPV) baculoviral DNA (10μg) in 950 μl transfection buffer (Ausubel et al., In Curr Protocols inMol. Biol. 1990). When the cells were cultured for 4 or 5 days, thetransfection supernatant contained recombinant viruses.

[0052] 3) Identification of Recombinant Baculovirus

[0053] The co-transfection lysates were diluted 10⁴, 10 ⁵ or 10⁶ andplated on Sf9 cells for cultivation for 4 to 6 days. After the plaqueswere well formed, plaques containing occlusion-negative cells wereidentified at a frequency of 1.3%. Several putative recombinant viralplaques were isolated and twice re-plaqued for purification. Purerecombinant viral plaque clones were isolated.

[0054] B. Analysis of Protein of Interest from Recombinant Baculovirus

[0055] 1) Preparation of Recombinant Virus Lysate

[0056] An insect cell Sf9 monolayer (2.5×10⁶ cells per 25-cm² flask) wasinfected with a recombinant virus clone and cultured in 5 ml GIBCOserum-free medium (from GIBCO Biochemicals, Rockville, Md.) or mediumsupplemented with 0.1% egg albumin to avoid contamination of serumbovine vitamin D binding protein. The culture flasks were incubated at27° C. and monitored daily for signs of infection. After 4 to 5 days,the cells were harvested by gently dislodging them from the flask andthe cells and culture medium were transferred to centrifuge tubes andcentrifuged for 10 min at 1000× g, 4° C. To maximize infection forrecombinant protein production, Sf9 cells were grown in a 100-ml spinnersuspension culture flask with 50 ml complete medium up to about 2×10⁶cells/ml. The cells were harvested, centrifuged at 1000× g for 10 minand re-suspended in 10 to 20 ml serum-free medium containing recombinantvirus at a multiplicity of infection (MOI) of 10. After 1 hour ofincubation at room temperature, the infected cells were transferred to a200-ml spinner flask containing 100 ml serum-free medium and incubatedfor 40 hr. More than 40% of the protein secreted was the protein ofinterest. The protein in the supernatant was isolated.

[0057] 2) Qualitative Estimation of the Protein of Interest

[0058] Coomassie Blue staining of the SDS-polyacrylamide gel, loading 20to 40 μg total cell protein per lane, was to estimate quantity ofexpressed protein. Because the samples contain cellular proteins, therecombinant protein was readily detected by comparison with uninfectedcellular proteins.

[0059] 3) Enzymatic Conversion of the Cloned Gc Protein to MacrophageActivating Factor (GcMAFc).

[0060] The cloned Gc protein (2 μg) with a molecular weight of 52,000and 458 amino acid residues (FIG. 3) was isolated by electroeluter andtreated with immobilized β-galactosidase and sialidase. The resultantcloned macrophage activating factor (GcMAFc) was added to mouse andhuman macrophages and assayed for phagocytic and superoxide generatingcapacity. Incubation of macrophages with 10 pg GcMAFc/ml for 3 hoursresulted in a 5-fold increased phagocytic and a 15-fold increase in thesuperoxide generating capacity of macrophages.

[0061] C. Subcloning of a Domain Required for Macrophage Activation

[0062] I. Cloning Procedure I: Non-fusion Vector.

[0063] 1) Cloning the Domain Responsible for Macrophage Activation(CdMAF)

[0064] The entire cDNA sequence for Gc protein in λgt11, including 76 bpof the upstream 5′ flanking region and 204 bp of the 3′ flankingstretch, was fragmented by EcoRi to yield four restriction fragmentsdesignated E1, 120; E2, 314; E3, 482; and E4, 748 bp, respectively. Eachwas cloned into the EcoRI site of the plasmid pSP65 from Promega(Madison, Wis.) by the method of Cooke and David (J. Clin. Invest., 762420, 1985). Although I found that a region less than one half of thedomain III was found to be responsible for macrophage activation, smallsegments less than 40 amino acid residues cannot be expressed in theinsect cells. Moreover, short peptides are rapidly degraded by proteasesin human plasma, and thus are not clinically useful. Accordingly, theentire domain III (approximately 80 amino acid residues) should besubcloned into an insect virus where I anticipate the efficientproduction and glycosylation of the peptide in the infected cells.

[0065] 2) Subcloning cDNA Fragment into the Polyhedron Gene ofBaculovirus.

[0066] Since the glycosylation of a peptide requires a leader signalsequence for transfer of the peptide into the endoplasmic reticulum, theDNA segment of E1 containing the initiation codon (−16 Met) through theleader sequence to the +1 amino acid (Leu) of the native Gc proteinshould be introduced into the vector. Because this segment carries theinitiation codon for the Gc protein, non-fusion vector, pVL1393(Invitrogen, San Diego, Calif.) was used. A segment containing theinitiation codon-leader sequence of the cDNA clone E1 and a segmentcoding for 85 C-terminal amino acids (the entire domain III plus 3′non-coding stretch) of the cDNA clone E4 were ligated together andcloned into the EcoRI site of the insect virus pVL vector. To achievethis construct, both E1 and E4 DNA were fragmented with HaeIII to yieldtwo fragments each; E1hl (87 bp), E1hs (33 bp) and E4hs (298 bp), E4hl(450 bp), respectively. Both the larger fragments E1hl and E4hl wereisolated electrophoretically, mixed with EcoRI-cut pVL, and ligated withT4 ligase, as shown in FIG. 4. This construct in correct orientationshould express the entire domain III, a total of 89 amino acids,including the 4 amino acids of E1hl, also referred to herein as CdMAF₁as shown in FIG. 5. To obtain the correct construction, competent E.coli HB101 cells are transformed with pVL vector and selected fortransformants on LB/ampicillin plates. DNA was prepared for sequencingprocedures to determine which colony contains the construct with properreading orientation by first searching for the 3′ poly dA stretch. Thoseclones with 3′ poly dA (from the poly A tail of mRNA) were thensequenced from the 5′ end to confirm correct orientation of the E1hlfragment. I found that the vector contains the entire construct (domainIII) in the correct orientation.

[0067] 3) Isolation of Recombinant Baculovirus, Purification of theCloned Domain Peptide (Cd) and Enzymatic Generation of the ClonedMacrophage Activating Factor (CdMAF)

[0068] Monolayers (2.5×10⁶ cells in each of 25-cm² flasks) of Spodopterafrugiperda (Sf9) cells were co-transfected with cloned plasmid DNA (2μg) and wild-type (AcMNPV) baculoviral DNA (10 μg) in 950 μltransfection buffer. Recombinant baculovirus plaques were isolated andused for production of the Gc domain III peptide in insect cells. Thiscloned domain with a molecular weight (MW) of 10,000 and 89 amino acidsas shown in FIG. 5, was purified electrophoretically. Two μg of thecloned domain (Cd) peptide was treated with immobilized β3-galactosidaseand sialidase to yield a cloned macrophage activating factor, designatedas CdMAF₁.

[0069] II. Cloning Procedure II: Fusion Vector.

[0070] 1) Cloning the Domain Responsible for Macrophage Activation(CdMAF)

[0071] A baculovirus fusion vector, pPbac vector (Stratagene, La Jolla,Calif.), contains human placental alkaline phosphatase secretory signalsequences that direct the nascent cloned peptide chain toward thesecretory pathway of the cells leading to secretion into culture media.The signal sequence is cleaved off by signal-sequence peptidase as thenascent cloned peptide is channeled toward the secretory pathway of thehost insect cells leading to secretion of the cloned domain (Cd)peptide. FIG. 6 depicts that the vector carries the stuffer fragment forgene substitution and lacZ gene for identification of the geneinsertion.

[0072] The stuffer fragment of pPbac vector was excised by digesting thevector DNA with restriction enzymes Smal and BamHI and was removed byelectroelution. The E4 cDNA fragment of the Gc protein was digested withHaeIII and BamHI, yielding a fragment practically the same as E4hl (seeFIG. 4). This fragment was mixed with the above pPbac vector and ligatedwith T4 ligase. This strategy not only fixes the orientation of ligationbut also fuses the fragment with the reading frame. The E. coli DH5aF′cells were transformed with the reaction mixture. The cloned DNA insertwas isolated from a number of colonies after digestion with HaeIII andBamHI. The insert was confirmed by sequencing. The sequence confirmedthe correct orientation.

[0073] 2) Isolation of Recombinant Baculovirus by Transfection of Sf9Insect Cells with Wild Type Baculovirus and the Cloned DNA Insert.

[0074] For transfection of insect cells (Spodoptera frugiperda, Sf9),linear wild type (AcMNPV) baculoviral DNA and insectin liposomes(Invitrogen, San Diego, Calif.) have been used. Liposome-mediatedtransfection of insect cells is the most efficient transfection methodavailable. For transfection to a monolayer of Sf9 cells (2×10⁶) in a 60mm dish a mixture of the following was gently added:

[0075] 3 μg cloned plasmid DNA

[0076] 10 μl linear wild type baculovirus (AcMNPV) DNA (0.1 μg/μl)

[0077] 1 ml medium

[0078] 29 μl insectin liposomes

[0079] The dishes were incubated at room temperature for 4 hours withslow rocking. After transfection, the 1 ml of medium was added andincubated at 27° C. in a humidified environment for 48 hours. Theresultant transfection lysate was plaque assayed. Purification ofrecombinant virus, isolation of the cloned domain peptide (Cd) andenzymatic generation of the cloned macrophage activating factordesignated CdMAF₂ were described in the Cloning Procedure I. This CdMAFis composed of 94 amino acid residues as shown in FIG. 7, including 9amino acids from the fusion vector and is referred to herein as CdMAF₂.Although CdMAF₂ has five amino acids more than the CdMAF₁ peptidederived from the non-fusion vector, they exhibited the same biologicalactivities.

Supporting Observations

[0080] A. Effects of Cloned Macrophage Activating Factors, GcMAFc andCdMAF on Cultured Phagocytes (Macrophages and Osteoclasts).

[0081] The three hour treatment of human macrophages and osteoclastswith picogram quantities (pg) of the cloned macrophage activatingfactors, GcMAFc and CdMAF, resulted in a greatly enhanced superoxidegenerating capacity of the phagocytes as shown in Table 1. The levels ofthe phagocyte activation are similar to those of macrophage activationby GcMAF (Yamamoto et al., AIDS Res. Human Ret. 11:1373, 1995). TABLE 1Activation of phagocytes by in vitro treatment with GcMAF and its clonedderivatives. nmole of superoxide produced/min/10⁶ phagocytes Conc. Mouseperitoneal Human pg/ml Human macrophages* macrophages osteoclasts GcMAFc 0 0.07 0.06 0.03  10 3.20 3.46 2.56 100 5.18 5.08 4.22 CdMAF  0 0.010.02 0.08  10 2.96 2.87 2.43 100 4.26 4.53 4.09

[0082] B. Activation of Mouse Peritoneal Macrophages by Administrationof Cloned Macrophage Activating Factors, GcMAFc and CdMAF.

[0083] One day post-administration of a picogram quantity (10 and 100pg/mouse) of GcMAFc or CdMAF to BALB/c mice, peritoneal macrophages wereisolated and assayed for superoxide generating capacity. As shown inTable 2, the macrophages were efficiently activated. These results aresimilar to those of macrophage activation with GcMAF (Naraparaju andYamamoto, Immunol. Lett. 43:143, 1994; Yamamoto et al., AIDS Res. HumanRet. 11:1373,1995). TABLE 2 Activation of mouse peritoneal macrophagesby administration of cloned GcMAF derivatives. Dosage nmole ofsuperoxide produced/min/10⁶ phagocytes pg/mouse Mouse peritonealmacrophages GcMAFc  0 0.05  10 3.18 100 5.23 CdMAF  0 0.03  10 2.54 1004.23

[0084] C. Therapeutic Effects of GcMAF, GcMAFc or CdMAF on Tumor BearingMice and Osteopetrotic Mice.

[0085] 1) Therapeutic Effects of GcMAF, GcMAFc or CdMAF on EhrlichAscites Tumor Bearing Mice.

[0086] When BALB/c mice were administered with GcMAF, GcMAFc or CdMAF(100 pg/mouse) and received 10⁵ Ehrlich ascites tumor cells/mouse, theysurvived for at least 5 weeks. All the control mice received only theascites tumor and died in approximately 14 days. When mice wereadministered with an additional 100 pg GcMAF/mouse 4 dayspost-transplantation, the tumor cells were completely eliminated (Table3).

[0087] When mice were transplanted with 10⁵ Ehrlich ascites tumorcells/mouse and treated twice with GcMAF, GcMAFc or CdMAF (100 pg/mouse)at 4 days and 8 days post-transplantation, all treated mouse groupssurvived over 65 days while the untreated 8 mouse groups all died atapproximately 13 days (Groups 4 through 9 of Table 3). TABLE 3Therapeutic effects of GcMAF and cloned derivatives on mice bearingEhrlich ascites tumor. No. of Post-transplantation No. of mice Groupmice treatment survived/period Group 1.  6 mice untreated control 6mice/13 ± 3 days 10 mice day 0 100 pg GcMAF/mouse 10 mice/36 ± 7 daysGroup 2.  6 mice untreated control 6 mice/14 ± 4 days 10 mice day 0 100pg GcMAFc/mouse 10 mice/35 ± 6 days Group 3.  6 mice untreated control 6mice/14 ± 5 days 10 mice day 0 100 pg CdMAF/mouse 10 mice/34 ± 3 daysGroup 4.  8 mice untreated control 8 mice/15 ± 5 days 12 mice day 0 100pg GcMAF/mouse day 4 100 pg GcMAF/mouse 12 mice/>65 days Group 5.  8mice untreated control 8 mice/14 ± 5 days 12 mice day 0 100 pgGcMAFc/mouse day 4 100 pg GcMAFc/mouse 12 mice/>65 days Group 6.  8 miceuntreated control 8 mice/14 ± 5 days 12 mice day 0 100 pg CdMAF/mouseday 4 100 pg CdMAF/mouse 12 mice/>65 days Group 7.  8 mice untreatedcontrol 8 mice/14 ± 4 days  8 mice day 4 100 pg GcMAF/mouse day 8 100 pgGcMAF/mouse 8 mice/>65 days Group 8.  8 mice untreated control 8 mice/13± 3 days  8 mice day 4 100 pg GcMAFc/mouse day 8 100 pg GcMAFc/mouse 8mice/>65 days Group 9.  8 mice untreated control 8 mice/13 ± 5 days  8mice day 4 100 pg CdMAF/mouse day 8 100 pg CdMAF/mouse 8 mice/>65 days

[0088] With respect to the results of Table 3, GcMAF was administeredintraperitoneally for Groups 1 through 6, and GcMAF was administeredintramuscularly (systemically) for Groups 7 through 9; mice in allgroups received 105 tumor cells/mouse.

[0089] 2) Therapeutic Effects of GcMAF and Cloned GcMAF Derivatives(GcMAFc and CdMAF) On Osteopetrotic Mice.

[0090] Administration of GcMAFc or CdMAF to new born litters ofosteopetrotic op/op mouse was performed by the weekly injection of 100picograms for four weeks beginning from a day after birth. Mice weresacrificed at 28 days. The tibiae were removed from the treated anduntreated control mice, longitudinally bisected, and examined under adissecting microscope to measure the size of the bone marrow cavity. Thecavity size was expressed as a percentage of the distance between theepiphyseal plates of the tibia. The untreated mouse group formed bonemarrow with 30% of the total length of tibia. The treated mouse groupexperienced a 20% increased bone marrow formation over that of theuntreated mouse group. This increased bone marrow cavity formation is anindication of osteoclast activation and increased osteoclastic boneresorption.

[0091] D. Therapeutic Effects of GcMAF, GcMAFc and CdMAF on Human Cancerand Virus Infected Patients.

[0092] 1. Cancer Patients: Therapeutic Effect of GcMAF on Prostate,Breast and Colon Cancer and Adult Leukemia Patients.

[0093] The administration of GcMAF (100 and 500 ng/human) to healthyvolunteers resulted in the greatly enhanced activation of macrophages asmeasured by the 7-fold enhanced phagocytic capacity and the 15-foldsuperoxide generating capacity of macrophages. The administration ofGcMAF showed no signs of any side effects to the recipients.Administration of various doses (100 pg to 10 ng/mouse) to a number ofmice produced neither ill effects nor histological changes in variousorgans including liver, lung, kidney, spleen, brain, etc. When patientswith various types of cancer were treated with GcMAF (100 ng/week),remarkable curative effects on various types of cancer were observed.The therapeutic efficacy of GcMAF on patients bearing various types ofcancers was assessed by tumor specific serum α-N-acetylgalactosaminidaseactivity because the serum enzyme level is proportional to the totalamount of cancerous cells (tumor burden). Curative effects of GcMAF onprostate, breast and colon cancer and leukemia are illustrated in FIGS.8A to 8D. After 25 weekly administrations of 100 ng GcMAF the majority(>90%) of prostate and breast cancer patients exhibited insignificantlylow levels of the serum enzyme. A similar result was also observed after35 GcMAF administrations to colon cancer patients. Similar curativeeffects of GcMAF on lung, liver, stomach, brain, bladder, kidney,uterus, ovarian, larynx, esophagus, oral and skin cancers were observed.Thus, GcMAF appeared to be effective on a variety of cancersindiscriminately. However, GcMAF showed no evidence of side effects inpatients after more than 6 months of therapy. This was also confirmed byblood cell counts profile, liver and kidney functions, etc.

[0094] When GcMAFc (100 ng/week) and CdMAF (100 ng/week) wereadministered to two prostate cancer patients each, curative effectssimilar to those of GcMAF were observed.

[0095] 2. Virus Infected Patients

[0096] Treatment of peripheral blood macrophages of HIV-infected/AIDSpatients with 100 pg GcMAF/ml resulted in a greatly enhanced macrophageactivation (Yamamoto et al., AIDS Res. Human Ret. 11:1373, 1995).HIV-infected patients carry anti-HIV antibodies. HIV-infected cellsexpress the viral antigens on the cell surface. Thus, macrophages have apotential to eliminate the infected cells via Fc-receptor mediatedcell-killing/ingestion when activated.

[0097] Similarly, treatment of peripheral blood macrophages of patentschronically infected with Epstein-Barr virus (EBV) and with herpeszoster with 100 ng GcMAF/ml resulted in a greatly enhanced macrophageactivation. Like HIV, EBV infects lymphocytes (B cells). Since theseenveloped viruses code for α-N-acetylgalactosaminidase and infectedcells secrete it into blood stream. Thus this enzyme activity in patientsera can be used as a prognostic index during therapy. Afterapproximately 25 administrations of GcMAF (100 ng/week) to patientschronically infected with EBV and with herpes zoster, the enzymeactivity decreased to that of healthy control levels. When GcMAFc (100ng/week) and CdMAF (100 ng/week) were administered to EBV-infectedpatients, curative effects similar to those of GcMAF were observed.

[0098] E. Adjuvant Activities of GcMAF, GcMAFc and CdMAF forImmunization and Vaccinations.

[0099] 1. Rapid Increase of the Number of Antibody Secreting Cells (PFC)in Mice after Administration of GcMAF and Sheep Erythrocytes.

[0100] BALB/c mice were inoculated with SRBC 6 hours after theintraperitoneal administration of 50 pg GcMAF/mouse. At variousintervals (1-5 days) after immunization, IgM-antibody secreting cells inthe spleen were determined using the Jerne plaque assay (Jerne et al.,Cell-bound antibodies, Wistar Institute Press, 1963). One daypost-administration of GcMAF and SRBC produced 1.35×10⁴ PFC/spleen. Twodays after administration of GcMAF and SRBC, the number of antibodysecreting cells had increased to 8.2×10⁴ PFC/spleen. By the 4th day thenumber of antibody secreting cells reached the maximal level (about23.6×10⁴ PFC/spleen), as shown in Table 4. In contrast, mice thatreceived an injection of SRBC alone produced about 3.8×10⁴ PFC/spleen, 4days after SRBC-injection.

[0101] To ascertain the dose response, mice were injected with SRBC 6hours after administration of various doses of GcMAF ranging from 1 to50 pg/mouse. On the 4th day post-administration of GcMAF and SRBC, thenumber of antibody secreting cells per spleen was determined by theJerne plaque assay. On the 4th day post-administration there was acommensurate increase in the number of plaque forming cells as theconcentration of GcMAF was increased above 1 pg per mouse. At a GcMAFdose of 5, 10 and 50 pg/mouse, I detected 12.6×10⁴, 20.2×10⁴ and24.3×10⁴ PFC/spleen, respectively. TABLE 4 Time course studies ondevelopment of cells secreting antibody against sheep erythrocytes(SRBC) in BALB/c mice after administration of GcMAF and SRBC^(a). AfterSRBC immunization Antibody secreting cells/spleen (×10⁴) (days) SRBConly GcMAF + SRBC 1  0.01 ± 0.002  1.35 ± 0.21 2 0.08 ± 0.02  8.28 ±1.26 3 1.18 ± 0.42 14.42 ± 2.32 4 3.86 ± 0.95 23.68 ± 6.05 5 2.15 ± 0.6318.63 ± 3.43

[0102] Without further elaboration the foregoing will so fullyillustrate my invention that others may, by applying current or futureknowledge, adapt the same for use under various conditions of service.

REFERENCES CITED

[0103] The following references are cited and their entire text isincorporated fully herein as are all references set forth above in thespecification.

U.S. Patent Documents

[0104] U.S. Pat. Nos. 5,177,001, 5,177,002 and 5,326,749 (Yamamoto).

Other Publications

[0105] 1. Jerne, N. K., Nordin, A. A. and Henry, C., The agar plaquetechnique for recognizing antibody producing cells. In Amos andKoprowski (eds). Cell-bound Antibody. Wistar Institute Press,Philadelphia, PA (1963).

[0106] 2. Sato, M., Tanaka, H., Yamada, T. and Yamamoto, N., Persistentinfection of BHK/WI-2 cells with rubella virus and characterization ofrubella variants. Arch. Virology 54:333-343 (1977).

[0107] 3. Reeves, J. D., August, C. S., Humbert, J. R., Weston, W. L,Host defense in infantile osteopetrosis. Pediatrics. 64:202-(1979).

[0108] 4. Marks, S. C., Jr., Congenital osteopetrotic mutations asprobes of the origin, structure and function of osteoclasts. Clin.Orthop. 189:239-(1984).

[0109] 5. Carbonell, L. F., Klowden, M. J. and Miller, L. K.,Baculovirus-mediated expression of bacterial genes in dipteran andmammalian cells. J. Virol. 56:153-160 (1985).

[0110] 6. Ngwenya, B. Z., and Yamamoto, N., Activation of peritonealmacrophages by lysophosphatidylcholine. Biochem. Biophys. Acta 839: 9-15(1985).

[0111] 7. Cooke, N. E. and Haddad, J. G., Vitamin D binding protein(Gc-globulin). Endocrine Rev. 10:294-307 (1989).

[0112] 8. Marks, S. C., Jr., Osteoclast biology: Lessons from mammalianmutations. Am. J. Med. Genet. 34:43-54 (1989).

[0113] 9. Ngwenya, B. Z. and Yamamoto, N., Contribution oflysophosphatidylcholine treated nonadherent cells to mechanism ofmacrophage stimulation. Proc. Soc. Exp. Biol. Med. 193:118-124 (1990).

[0114] 10. Ausubel, F. A., Brent, R., Kingston, R. E., Moore, D. D.,Seidman, J. G., Smith, J. A. and Struhl, K. (eds.), Expression ofproteins in insect cells using baculoviral vectors. Current Protocols inMolecular Biology. Sections 16.8.1-16.11.7. Greene Publishing andWiley-Interscience, New York (1990).

[0115] 11. Yagi, F., Eckhardt, A. E. and Goldstein I. J., Glycosidasesof Ehrlich ascites tumor cells and ascitic fluid-purification andsubstrate specificity of α-N-acetylgalactosaminidase andα-galactosidase: comparison with coffee bean α-galactosidase. Arch.Biochem. Biophys. 280:61-67 (1990).

[0116] 12. Yamamoto, N. and Homma, S., Vitamin D₃ binding protein(group-specific component, Gc) is a precursor for the macrophageactivating signal from lysophosphatidylcholine-treated lymphocytes.Proc. Natl. Acad. Sci. USA. 88:8539-8543 (1991).

[0117] 13. Cooke, N. E. and David, E. V., Serum vitamin D-bindingprotein is a third member of the albumin and alpha-fetoprotein genefamily. J. Clin. Invest. 76:2420-2424 (1985).

[0118] 14. Haddad, J. G., Hu, Y. Z., Kowalski, M. A., Laramore, C., Ray,K., Robzyk, P. and Cooke, N. E., Identification of the sterol- andactin-binding domains of plasma vitamin D binding protein (Gc-globulin).Biochemistry 31:71747181 (1992).

[0119] 15. Yamamoto, N. and Kumashiro, R., Conversion of vitamin D₃binding protein (Group-specific component) to a macrophage activatingfactor by stepwise action of β-galactosidase of B cells and sialidase ofT cells. J. Immunol. 151:27-94-2902 (1993).

[0120] 16. Homma, S., Yamamoto, M. and Yamamoto, N., Vitamin D bindingprotein (group-specific component, Gc) is the sole serum proteinrequired for macrophage activation after treatment of peritoneal cellswith lysophosphatidylcholine. Immunol. Cell Biol. 71:249-257 (1993).

[0121] 17. Yamamoto, N., Kumashiro, R., Yamamoto, M., Willett, N. P. andUndsay, D. D., Regulation of inflammation-primed activation ofmacrophages by two serum factors, vitamin D₃-binding protein andalbumin. Inf. Imm. 61:5388-5391 (1993).

[0122] 18. Yamamoto, N., Lindsay, D. D., Naraparaju, V. R., Irelalnd, R.A. and Popoff, S. M., A defect in the inflammation-primed macrophageactivation cascade in osteopetrotic (op) rats. J. Immunol. 152:5100-5107(1994).

[0123] 19. Yamamoto, N., Willett, N. P. and Lindsay, D. D.,Participation of serum proteins in the inflammation-primed activation ofmacrophages. Inflammation. 18:311-322 (1994).

[0124] 20. Naraparaju, V. R. and Yamamoto, N., Roles of β-galactosidaseof B lymphocytes and sialidase of T lymphocytes in inflammation-primedactivation of macrophages. Immunol. Lett. 43:143-148 (1994).

[0125] 21. Yamamoto, N., Naraparaju, V. R. and Srinivasula, S. M.,Structural modification of serum vitamin D₃-binding protein andimmunosuppression in HIV-infected patients. AIDS Res. Human Ret.11:1373-1378 (1995).

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 3(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 458 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii)MOLECULE TYPE: protein (iii) HYPOTHETICAL: no (vi) ORIGINAL SOURCE: (A)ORGANISM: Human (C) INDIVIDUAL ISOLATE: Vitamin D-binding protein (Gcprotein) (x) PUBLICATION INFORMATION: (A) AUTHORS: Cooke, Nancy E.,David, E Vivek (B) TITLE: Serum Vitamin D-binding Protein is a ThirdMember of the Albumin and Alpha Fetoprotein Gene Family (C) JOURNAL: J.Clinical Investigation (D) VOLUME: 76 (E) ISSUE: 12 (F) PAGES: 2420-2424(G) DATE: December, 1985 (K) RELEVANT RESIDUES IN SEQ ID NO:1: FROM1-485 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Leu Glu Arg Gly Arg AspTyr Glu Lys Asn Lys Val Cys Lys Glu Phe 5 10 15 Ser His Leu Gly Lys GluAsp Phe Thr Ser Leu Ser Leu Val Leu Tyr 20 25 30 Ser Arg Lys Phe Pro SerGly Thr Phe Glu Gln Val Ser Gln Leu Val 35 40 45 Lys Glu Val Val Ser LeuThr Glu Ala Cys Cys Ala Glu Gly Ala Asp 50 55 60 Pro Asp Cys Tyr Asp ThrArg Thr Ser Ala Leu Ser Ala Lys Ser Cys 65 70 75 80 Glu Ser Asn Ser ProPhe Pro Val His Pro Gly Thr Ala Glu Cys Cys 85 90 95 Thr Lys Glu Gly LeuGlu Arg Lys Leu Cys Met Ala Ala Leu Lys His 100 105 110 Gln Pro Gln GluPhe Pro Thr Tyr Val Glu Pro Thr Asn Asp Glu Ile 115 120 125 Cys Glu AlaPhe Arg Lys Asp Pro Lys Glu Tyr Ala Asn Gln Phe Met 130 135 140 Trp GluTyr Ser Thr Asn Tyr Glu Gln Ala Pro Leu Ser Leu Leu Val 145 150 155 160Ser Tyr Thr Lys Ser Tyr Leu Ser Met Val Gly Ser Cys Cys Thr Ser 165 170175 Ala Ser Pro Thr Val Cys Phe Leu Lys Glu Arg Leu Gln Leu Lys His 180185 190 Leu Ser Leu Leu Thr Thr Leu Ser Asn Arg Val Cys Ser Gln Tyr Ala195 200 205 Ala Tyr Gly Glu Lys Lys Ser Arg Leu Ser Asn Leu Ile Lys LeuAla 210 215 220 Gln Lys Val Pro Thr Ala Asp Leu Glu Asp Val Leu Pro LeuAla Glu 225 230 235 240 Asp Ile Thr Asn Ile Leu Ser Lys Cys Cys Glu SerAla Ser Glu Asp 245 250 255 Cys Met Ala Lys Glu Leu Pro Glu His Thr ValLys Leu Cys Asp Asn 260 265 270 Leu Ser Thr Lys Asn Ser Lys Phe Glu AspCys Cys Gln Glu Lys Thr 275 280 285 Ala Met Asp Val Phe Val Cys Thr TyrPhe Met Pro Ala Ala Gln Leu 290 295 300 Pro Glu Leu Pro Asp Val Arg LeuPro Thr Asn Lys Asp Val Cys Asp 305 310 315 320 Pro Gly Asn Thr Lys ValMet Asp Lys Tyr Thr Phe Glu Leu Ser Arg 325 330 335 Arg Thr His Leu ProGlu Val Phe Leu Ser Lys Val Leu Glu Pro Thr 340 345 350 Leu Lys Ser LeuGly Glu Cys Cys Asp Val Glu Asp Ser Thr Thr Cys 355 360 365 Phe Asn AlaLys Gly Pro Leu Leu Lys Lys Glu Leu Ser Ser Phe Ile 370 375 380 Asp LysGly Gln Glu Leu Cys Ala Asp Tyr Ser Glu Asn Thr Phe Thr 385 390 395 400Glu Tyr Lys Lys Lys Leu Ala Glu Arg Leu Lys Ala Lys Leu Pro Glu 405 410415 Ala Thr Pro Thr Glu Leu Ala Lys Leu Val Asn Lys Arg Ser Asp Phe 420425 430 Ala Ser Asn Cys Cys Ser Ile Asn Ser Pro Pro Leu Tyr Cys Asp Ser435 440 445 Glu Ile Asp Ala Glu Leu Lys Asn Ile Leu 450 455 458 (2)INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:89 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (iii) HYPOTHETICAL: no (vi) ORIGINAL SOURCE: (A) ORGANISM:Human (C) INDIVIDUAL ISOLATE: Vitamin D-binding protein (Gc protein) (x)PUBLICATION INFORMATION: (A) AUTHORS: Cooke, Nancy E., David, E Vivek(B) TITLE: Serum Vitamin D-binding Protein is a Third Member of theAlbumin and Alpha Fetoprotein Gene Family (C) JOURNAL: J. ClinicalInvestigation (D) VOLUME: 76 (E) ISSUE: 12 (F) PAGES: 2420-2424 (G)DATE: December, 1985 (K) RELEVANT RESIDUES IN SEQ ID NO:2: FROM 1 TO 4and 5 TO 89 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Leu Glu Arg Gly ProLeu Leu Lys Lys Glu Leu Ser Ser Phe Ile Asp 5 10 15 Lys Gly Gln Glu LeuCys Ala Asp Tyr Ser Glu Asn Thr Phe Thr Glu 20 25 30 Tyr Lys Lys Lys LeuAla Glu Arg Leu Lys Ala Lys Leu Pro Glu Ala 35 40 45 Thr Pro Thr Glu LeuAla Lys Leu Val Asn Lys Arg Ser Asp Phe Ala 50 55 60 Ser Asn Cys Cys SerIle Asn Ser Pro Pro Leu Tyr Cys Asp Ser Glu 65 70 75 80 Ile Asp Ala GluLeu Lys Asn Ile Leu 85 89 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 94 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: no (vi)ORIGINAL SOURCE: (A) ORGANISM: Human (C) INDIVIDUAL ISOLATE: VitaminD-binding protein (Gc protein) (x) PUBLICATION INFORMATION: (A) AUTHORS:Cooke, Nancy E., David, E Vivek (B) TITLE: Serum Vitamin D-bindingProtein is a Third Member of the Albumin and Alpha Fetoprotein GeneFamily (C) JOURNAL: J. Clinical Investigation (D) VOLUME: 76 (E) ISSUE:12 (F) PAGES: 2420-2424 (G) DATE: December, 1985 (K) RELEVANT RESIDUESIN SEQ ID NO:3: FROM 10 TO 94 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:Ile Ile Pro Val Glu Glu Glu Asn Pro Pro Leu Leu Lys Lys Glu Leu 5 10 15Ser Ser Phe Ile Asp Lys Gly Gln Glu Leu Cys Ala Asp Tyr Ser Glu 20 25 30Asn Thr Phe Thr Glu Tyr Lys Lys Lys Leu Ala Glu Arg Leu Lys Ala 35 40 45Lys Leu Pro Glu Ala Thr Pro Thr Glu Leu Ala Lys Leu Val Asn Lys 50 55 60Arg Ser Asp Phe Ala Ser Asn Cys Cys Ser Ile Asn Ser Pro Pro Leu 65 70 7580 Tyr Cys Asp Ser Glu Ile Asp Ala Glu Leu Lys Asn Ile Leu 85 90 94

I claim:
 1. A process for cloning vitamin D₃-binding protein (Gcprotein) into baculovirus comprising the step of selecting and using abaculovirus vector to clone the vitamin D₃-binding protein Gc protein(Gc protein).
 2. A process for producing a cloned macrophage activatingfactor (GcMAFc) comprising contacting cloned Gc protein in vitro withimmobilized β-galactosidase and sialidase and obtaining the clonedmacrophage activating factor (GcMAFc).
 3. A process for cloning vitaminD₃-binding protein domain III (Gc domain III) into baculoviruscomprising the step of selecting and utilizing a baculovirus vector toclone the vitamin D₃-binding protein domain III (Gc domain III).
 4. Aprocess for producing a cloned macrophage activating factor (CdMAF)comprising contacting cloned Gc domain III in vitro with immobilizedβ-galactosidase and sialidase and obtaining the macrophage activatingfactor (CdMAF).
 5. A method of treating a person suffering from cancerby administering to the person a therapeutically effective amount of aGc protein macrophage activating factor (GcMAF), the GcMAF being aproduct of contacting serum Gc protein in vitro with immobilizedβ-galactosidase and sialidase.
 6. A method of treating a personsuffering from cancer by administering to the person a therapeuticallyeffective amount of a cloned macrophage activating factor (GcMAFc),which is a product of the process according to claim
 2. 7. A method oftreating a person suffering from cancer by administering to the person atherapeutically effective amount of a cloned macrophage activatingfactor (CdMAF), which is a product of the process according to claim 4.8. A method of treating a person suffering from human immunodeficiencyvirus (HIV), Epstein-Barr virus (EBV) or herpes zoster by administeringto the person a therapeutically effective amount of a macrophageactivating factor (GcMAF), which is a product of contacting serum Gcprotein in vitro with immobilized β-galactosidase and sialidase.
 9. Amethod of treating a person suffering from human immunodeficiency virus(HIV), Epstein-Barr virus (EBV) or herpes zoster by administering to theperson a therapeutically effective amount of a macrophage activatingfactor (GcMAFc), which is a product of the process according to claim 2.10. A method of treating a person suffering from human immunodeficiencyvirus (HIV), Epstein-Barr virus (EBV) or herpes zoster by administeringto the person a therapeutically effective amount of a macrophageactivating factor (CdMAF), which is a product of the process accordingto claim
 4. 11. A macrophage activating factor (GcMAFc), which is aproduct of the process according to claim
 2. 12. A macrophage activatingfactor (CdMAF), which is a product of the process according to claim 4.13. A method of promoting bone marrow formation in osteopetroticpatients comprising administering a therapeutically effective amount ofa macrophage activating factor (GcMAFc), which is a product of theprocess according to claim
 2. 14. A method of promoting bone marrowformation in osteopetrotic patients comprising administering atherapeutically effective amount of a macrophage activating factor(CdMAF), which is a product of the process according to claim
 4. 15. Anadjuvant for immunizing humans and animals with antigens or vaccines,the adjuvant comprising a macrophage activating factor (GcMAF) which isa product of contacting serum Gc protein in vitro with immobilizedβ-galactosidase and sialidase.
 16. An adjuvant for immunizing humans andanimals with antigens or vaccines, the adjuvant comprising a macrophageactivating factor (GcMAFc), which is a product of the process accordingto claim
 2. 17. An adjuvant for immunizing humans and animals withantigens or vaccines, the adjuvant comprising a macrophage activatingfactor (CdMAF), which is a product of the process according to claim 4.18. A cloned vitamin D₃-binding protein (Gc protein) having an aminoacid sequence of FIG. 3 (SEQ. ID. NO:1)(GcMAFc).
 19. A cloned vitaminD₃-binding protein domain III (Gc domain III) having an amino acidsequence of FIG. 5 (SEQ ID. NO:2)(CdMAF₁).
 20. A cloned vitaminD₃-binding protein domain III (Gc domain II) having an amino acidsequence of FIG. 7 (SEQ ID. NO:3)(CdMAF₂).